Background: Sick sinus syndrome (SSS) is common and may require electronic pacemaker implantation, gene therapy (biopacemaking) may be an alternative. For SSS, repair may be better than the generation of a de novo biopacemaker, due to the complex nature of the sinoatrial node (SAN). We hypothesised that an ex vivo model of SSS could be created by the identification of a subsidiary pacemaker in the SAN region, and that expression of a pacemaker channel (HCN4 or HCN212) in this region would accelerate the pacing rate. Methods: A bradycardic SSS model was generated by the removal of the upper two thirds of a rat SAN and a system to record the intrinsic activity during tissue culture was developed. The leading pacemaker site of the SSS preparations were identified by activation mapping then characterised by If blockade, β-adrenergic stimulation, histology and immunohistochemistry. Further SSS preparations were injected in this region with recombinant adenovirus (RAd) expressing no functional ion channel (Ad5-GFP or Ad5-GFP-HCN4Δ); or RAd expressing a functional If channel (Ad5-HCN212 or Ad5-PREK-HCN4). During tissue culture electrical activity was monitored using bipolar electrodes. Results: Tissue culture and monitoring of the rat SAN is feasible and does not induce significant changes in HCN4 or connexin-43 expression. The uninjected SSS preparations displayed a slower rate than the control SAN (p<0.001). In 5/6 cases the subsidiary pacemaker was HCN4 -ve and Connexin-43 +ve (in contrast to the SAN) and was close to the superior aspect of the inferior vena cava. The cell size of the subsidiary pacemaker was comparable to that of the SAN and smaller than working myocardium (p<0.001). Pacing was responsive to β-adrenergic stimulation and was partially dependent on If current. The pacing rate of the AdHCN212-injected SSS preparations was significantly faster than the uninjected SSS preparations (p<0.001). The remaining RAd did not significantly affect the pacing rate of the SSS model. Conclusions: There is subsidiary pacemaker tissue close to the superior aspect of the IVC that shares some characteristics of the SAN. These results suggest that adenovirus-mediated expression of HCN channels in subsidiary pacemaker tissue of the right atrium may be a useful strategy in biopacemaking for SSS.